Methods, systems, and kits for intravascular nucleic acid delivery

ABSTRACT

Nucleic acid transfection of vascular smooth muscle cells is enhanced by the application of vibrational energy to the cells. By applying vibrational energy at frequency in the range from 1 kHz to 10 MHz and at an intensity in the range from 0.01 W/cm 2  to 100 W/cm 2 , significant enhancement of the uptake of nucleic acids into vascular smooth muscle cells can be achieved.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 10/087,179 (Attorney Docket No. 017148-0001220), filed Mar. 10,2002, which was a continuation of application Ser. No. 09/223,231(Attorney Docket No. 17148-001210) filed Dec. 30, 1998, now U.S. Pat.No. 6,372,498, which claimed the benefit of provisional applicationserial No. 60/070,073 (Attorney Docket No. 017148-001200), filed on Dec.31, 1997. The full disclosure of each is incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to medical methods anddevices. More particularly, the present invention relates to methods,systems, and kits for the delivery of nucleic acids to smooth musclecells which line the lumen of blood vessels.

[0004] A number of percutaneous intravascular procedures have beendeveloped for treating atherosclerotic disease in a patient'svasculature. The most successful of these treatments is percutaneoustransluminal angioplasty (PTA) which employs a catheter having anexpansible distal end, usually in the form of an inflatable balloon, todilate a stenotic region in the vasculature to restore adequate bloodflow beyond the stenosis. Other procedures for opening stenotic regionsinclude directional atherectomy, rotational atherectomy, laserangioplasty, stents and the like. While these procedures, particularlyPTA, have gained wide acceptance, they continue to suffer from thesubsequent occurrence of restenosis.

[0005] Restenosis refers to the re-narrowing of an artery within weeksor months following an initially successful angioplasty or other primarytreatment. Restenosis afflicts up to 50% of all angioplasty patients andresults at least in part from smooth muscle cell proliferation inresponse to the injury caused by the primary treatment, generallyreferred to as “hyperplasia.” Blood vessels in which significantrestenosis occurs will require further treatment.

[0006] A number of strategies have been proposed to treat hyperplasiaand reduce restenosis. Such strategies include prolonged ballooninflation, treatment of the blood vessel with a heated balloon,treatment of the blood vessel with radiation, the administration ofanti-thrombotic drugs following the primary treatment, stenting of theregion following the primary treatment, and the like. While enjoyingdifferent levels of success, no one of these procedures has proven to beentirely successful in treating all occurrences of restenosis andhyperplasia.

[0007] Of particular interest, it has recently been proposed to delivernucleic acids to smooth muscle cells within blood vessels for thetreatment of hyperplasia and other disease conditions. See, e.g. U.S.Pat. No. 5,328,470. Progress in vascular gene therapy, however, has beenhindered by the limited efficiency and/or toxicity of most currentlyavailable transfection materials and techniques. Current methods used toachieve nucleic acid transfer into vascular smooth muscle cells comprisethe delivery of naked DNA, cationic liposomes, and specializedadenoviral and retroviral vectors. Each of these approaches areproblematic. While the use of adenoviral vectors can achieve relativelyhigh transfection efficiencies, the use of viruses raises concern amongmany experts in the field.

[0008] For these reasons, it would be desirable to provide additionaland/or improved methods, systems, kits, and the like for the delivery ofnucleic acids to vascular smooth muscle cells and other cells whichcomprise the vascular wall. It would be particularly desirable if suchgene delivery methods were useful for the treatment of hyperplasia inregions of a blood vessel which have previously been treated byangioplasty, atherectomy, stenting, and other primary or secondarytreatment modalities for atherosclerotic disease. Such methods shouldprovide efficient gene delivery, result in minimum necrosis of the cellslining the vasculature (particularly smooth muscle cells and endothelialcells), permit targeting of vascular smooth muscle cells, be capable ofbeing performed with relatively simple catheters and other equipment,and suffer from minimum side effects. At least some of these objectiveswill be met by the invention described hereinafter.

[0009] 2. Description of the Background Art

[0010] Catheters and methods for intravascular transfections aredescribed in U.S. Pat. No. 5,328,470 and published in PCT applicationsWO 97/12519; WO 97/11720; WO 95/25807; WO 93/00052; and WO 90/11734.

[0011] Ultrasound-mediated cellular transfection is described orsuggested in Kim et al. (1996) Hum. Gene Ther. 7:1339-1346; Tata et al.(1997) Biochem. Biophy. Res. Comm. 234:64-67; and Bao et al. (1997)Ultrasound in Med. & Biol. 23:953-959. The effects of ultrasound energyon cell wall permeability and drug delivery are described in Harrison etal. (1996) Ultrasound Med. Biol. 22:355-362; Gao et al. (1995) GeneTher. 2:710-722; Pohl et al. (1993) Biochem. Biophys. Acta.1145:279-283; Gambihler et al. (1994) J. Membrane Biol. 141:267-275;Bommannan et al. (1992) Pharma. Res. 9:559-564; Tata and Dunn (1992) J.Phys. Chem. 96:3548-3555; Levy et al. (1989) J. Clin. Invest.83:2074-2078; Feschheimer et al. (1986) Eur. J. Cell Biol. 40:242-247;and Kaufman et al. (1977) Ultrasound Med. Biol. 3:21-25. A device andmethod for transfection, endothelial cells suitable for seeding vascularprostheses are described in WO 97/13849.

[0012] Local gene delivery for the treatment of restenosis followingintravascular intervention is discussed in Bauters and Isner (1998)Progr. Cardiovasc. Dis. 40:107-116 and in Baek and March (1998) Circ.Res. 82:295-305.

[0013] A high frequency ultrasonic catheter employing an air-backedtransducer which may be suitable for performing certain methodsaccording to the present invention is described in He et al. (1995) Eur.Heart J. 16:961-966. Other catheters suitable for performing at leastsome methods according to the present invention are described inco-pending Application Nos. 08/565,575; 08/566,740; 08/566,739;08/708,589; 08/867,007, and 09/ 09/223,225 (Attorney Docket No.17148-001400, filed on Dec. 30, 1998), assigned to the assignee of thepresent invention, the full disclosures of which are incorporated hereinby reference.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention comprises methods, systems, and kits forthe delivery of nucleic acids to the smooth muscle cells of the typewhich line coronary arteries and other blood vessels. The delivery ofnucleic acids to target cells is generally referred to as“transfection,” and the transfection methods of the present inventionare advantageous since they are capable of significantly increasingtransfection efficiency, i.e. the amount of nucleic acid materials takenup by the smooth muscle cells to which they are delivered. The methodsof the present invention are useful with a wide variety of nucleic acidtypes. For example, it has been found that significant transfectionefficiencies can be obtained even with naked DNA and RNA molecules i.e.,nucleic acids which are not incorporated into liposomes, viral vehicles,plasmids, or other conventional nucleic acid vehicles. The methods arenot limited to such naked nucleic acids, however, they are also suitablefor the delivery of nucleic acids incorporated into liposomes and othervesicles; viral vectors, including both adenoviral vectors andretroviral vectors; plasmids, and the like.

[0015] The methods of the present invention are particularly suitablefor delivering nucleic acids incorporated into liposomes often referredto as “lipofection,” to the vascular smooth muscle cells. As isdemonstrated in the Experimental section hereinafter, transfection ofvascular smooth muscle cells with naked DNA is enhanced significantly byvibratory energy (by a factor of 7.5 in the particular data shown), butoverall transfection efficiency still remains at a relatively low level.In contrast, lipofection enhanced with vibratory energy according to thepresent invention shows a lesser enhancement over lipofection withoutvibrational energy (by a factor of three in the particular data whichare shown), but the overall transfection efficiency, is substantiallygreater than that which can be achieved with naked nucleic acids, evenwith vibrational energy enhancement. Thus, the combination oflipofection with vibrational energy enhancement will frequently bepreferred. While similar overall transfection efficiencies may beachieved with vibrational enhancement of viral vectors, the use of viralvectors will often not be preferred because of the safety concerns whichhave been raised with respect to such delivery vehicles. Additionally,as other delivery vehicles are developed as alternatives for variationsof the liposome and viral vehicles which presently find use, it will beexpected that the vibratory enhancements of the present invention willfind use with such methods. A significant advantage of the presentinvention, however, is that such delivery vehicles are not essential forefficient uptake.

[0016] While the methods, systems, and kits of the present inventionwill preferably be used with in vivo transfection techniques describedabove, they will also find use with in vitro techniques for transfectingvascular smooth muscle cells in culture. Such in vitro methods will finduse in many contexts, such as in the testing of different structural andregulatory genes to determine their effect on vascular smooth musclecells, the transformation of vascular smooth muscle cells to otherpredictable phenotypes research purposes, and the like. In otherinstances, it may be desirable to transfect autologous or heterologousvascular smooth muscle cells in vitro so that the cells can later be“seeded” back into a patient for a particular therapeutic purpose. Forexample, vascular smooth muscle cells can be transfected to producetherapeutic proteins which can be released by the transfected cellsafter they are implanted or otherwise introduced to a patient.

[0017] The nucleic acids may be in the form of genes, gene fragments,sense oligonucleotides and polynucleotides, anti-sense oligonucleotidesand polynucleotides, and any other type of nucleic acid havingbiological activity or benefit. Exemplary genes that may be deliveredfor treating cardiovascular disease and hyperplasia include angiogenicfactors, such as vascular endothelial growth factor (VEGF), endothelialnitric oxide synthase (eNOS), tissue inhibitor matrixmatallio-proteinase (TIMP), p21, and the like.

[0018] Smooth muscle and other vascular cells are transfected accordingto the present invention by delivering nucleic acids to the cellslocated, for example, in a target region within a blood vessel or incell culture. The cells are exposed to vibratory energy at a frequencyand intensity selected to enhance the uptake of the nucleic acids by thesmooth muscle cells, which line the blood vessel wall. The exposure ofthe cells to the vibratory energy can occur before exposure orintroduction of the nucleic acids, after exposure or introduction of thenucleic acids, or simultaneously with such exposure or introduction.Preferably, exposure of the cells to the vibratory energy will continuefor at least a time (total elapsed time) following the introduction orexposure of the cells to the nucleic acids, typically for at least 10seconds, preferably for at least 60 seconds, more preferably for atleast 300 seconds, and still more preferably for at least 900 seconds,usually being in the range from 10 seconds to 900 seconds.

[0019] Preferably, the vibratory energy is delivered at a frequency inthe range from 1 kHz to 10 MHz, preferably in the range from 20 kHz to 3MHz, usually from 100 kHz to 2 MHz. The intensity of the vibrationalenergy will usually be in the range from 20 W/cm² to 100 W/cm²,preferably in the range from 0.1 W/cm² to 10 W/ cm², usually from 0.5W/cm² to 5 W/cm². The vibratory energy may be delivered continuouslyduring the transfection event, or alternatively may be deliveredintermittently, e.g. with a duty cycle within the range from 1% to 100%,usually from 5% to 95%, preferably from 10% to 50%.

[0020] The duration of exposure of the cells to the vibration energywill be a function of total elapsed time (usually within the range andlimits set forth above), the duty cycle (percentage of the total elapsedtime in which the vibrational energy is turned on), and pulse repetitionfrequency (PRF; the frequency at which the vibrational energy is turnedoff and on, typically in the range from 1 Hz to 1000 Hz). Generally, theduty cycle and/or PRF can be controlled to permit heat dissipation tomaintain a temperature in the treated artery or cell culture below 45°C., preferably below 42° C., and more preferably below 40° C. Highertemperatures can be deleterious to the viability of the vascular smoothmuscle cells.

[0021] The vibrational energy will usually be ultrasonic energy and maybe delivered in a variety of ways. For example, the vibrational energymay be delivered from an external source, e.g. by focused ultrasonicsystems, such as high intensity focused ultrasound (HIFU) systems whichare commercially available. Usually, however, the ultrasonic energy willbe delivered intravascularly using an interface surface which isdisposed within the region within the blood vessel. The interfacesurface is vibrationally excited to radiate ultrasonic energy directlyor indirectly (as defined below) into the blood vessel wall. Typically,the ultrasonic surface is carried on a flexible catheter having avibrational transducer or other oscillator disposed on the catheter nearthe surface. The transducer is then energized to vibrate the surfacewithin the desired frequency range and at the desired intensity.Alternatively, ultrasonic or other vibrational energy can be deliveredfrom an external source down a transmission member through the catheterto the interface surface. For in vitro methods, a variety of hand-heldprobes and transducers could be employed. A particular transducer usefulfor imparting vibratory energy to cultures of vascular smooth musclecells is described in the Experimental section hereinafter.

[0022] The vibrational energy may be delivered directly into the bloodvessel wall, e.g. by contacting the interface surface directly against aportion of the wall within the target region. Alternatively, thevibrational energy can be delivered indirectly by vibrating the surfacewithin blood or other liquid medium within the blood vessel. Usually,the nucleic acids will be released or disposed in the liquid medium. Inan exemplary embodiment, the nucleic acids are contained within asuitable transfection medium which is localized within the target regionby a pair of axially spaced-apart balloons. The interface surface isalso disposed between the balloons, and energy is applied to theentrapped medium via the interface surface. Alternatively, the nucleicacid medium may be delivered to the interior of a porous balloon and/orto fluid delivery conduits secured to the outside of a balloon, where inboth cases the vibrational transducer can be mounted on the catheterbody within the balloon. Conveniently, the medium containing the nucleicacids can be delivered to the region via the same catheter, optionallybeing recirculated or replenished via the catheter during the treatment.

[0023] Alternatively, the nucleic acids can be delivered to the patientsystemically while the vibrational energy is applied locally and/or froman external source as described above. Optionally, the nucleic acids maybe delivered to a vascular target site in the presence of microbubblesof gas or other cavitation nucleation components. It is believed thatlow intensity vibration of the type preferably employed in the methodsof the present invention will generally not induce cavitation in avascular environment devoid of cavitation nucleii. As cavitation ispresently believed to contribute to the formation of pores in the wallsof the smooth muscle cells (and thus enhance nucleic acid uptake), theintroduction of microbubbles or other cavitation nucleii together withthe nucleic acids, e.g. from the same delivery catheter, maysignificantly enhance the nucleic acid uptake. For example, the nucleicacids may be delivered in a liquid medium to which dissolved gases havebeen added as cavitation nucleii.

[0024] The nucleic acids can be delivered to the smooth muscle or othervascular cells for a variety of purposes. In a preferred example, thenucleic acids are delivered to a region of the blood vessel which haspreviously been treated by a primary intravascular technique fortreating cardiovascular disease, such as balloon angioplasty,directional atherectomy, rotational atherectomy, stenting, or the like.The methods of the present invention for inhibiting intimal hyperplasiain vascular smooth muscle cells will find particular use followingstenting procedures in order to prevent or inhibit hyperplasia which canoccur following stenting. The nucleic acids delivered will be intendedto inhibit hyperplasia and/or promote angiogenesis following suchprimary treatment. Methods for promoting angiogenesis, of course, neednot be performed in conjunction with a primary treatment. Suitable genesfor such treatments have been described above.

[0025] Kits according to the present invention will comprise a catheterhaving an interface surface which may be vibrated. The kits will furtherinclude instructions for use setting forth a method as described above.Optionally, the kits will further include packaging suitable forcontaining the catheter and the instructions for use. Exemplarycontainers include pouches, trays, boxes, tubes, and the like. Theinstructions for use may be provided on a separate sheet of paper orother medium. Optionally, the instructions may be printed in whole or inpart on the packaging. Usually, at least the catheter will be providedin a sterilized condition. Other kit components, such as the nucleicacids to be delivered, may also be included.

[0026] Systems according the present invention will comprise a catheterhaving an interface surface which may be vibrated at the frequencies andpower levels described above. Such systems may further include nucleicacids in a form suitable for the transfection or lipofection of vascularsmooth muscle cells lining an artery or other blood vessel. The nucleicacids may be naked, viral-associated, but will preferably beincorporated within liposome vesicles in order to enhance transfectionefficiency when delivered in the presence of vibratory energy accordingto the methods of the present invention. The catheter will usually bepackaged in a sterile tray, pouch, or other conventional container,while the nucleic acid reagent will be incorporated in an ampoule,bottle, or other conventional liquid pharmaceutical container.Optionally, the catheter and reagent will be packaged together in a box,bag, or other suitable package. Further optionally, the systems mayinclude instructions for use as described above.

[0027] In a particular aspect of the present invention, it has beenfound that the delivery of vibrational energy to the wall of a bloodvessel will enhance or increase the efficacy of gene expression by atleast two-fold, often at least four-fold, and preferably even greater.The data in the Experimental section hereinafter demonstrate that genedelivery to intravascular tissue in the presence of vibrational energyis enhanced four-fold when compared to gene delivery under the sameconditions in control blood vessels in an animal model. Such enhancementin gene delivery promises to significantly improve vascular therapieswhich rely on transfection of vascular tissues, such as the treatment ofrestenosis as described above. Moreover, it has been found that theultrasound conditions which successfully enhance transfection of bloodvessel tissues are also effective at directly inhibiting hyperplasia, asshown for example in U.S. Pat. No. 6,210,393, which is commonly assignedand has common inventorship with the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a perspective view of a catheter suitable for use in themethods of the present invention.

[0029]FIG. 2 is a cross-sectional view taken along line 2-2 of FIG. 1.

[0030]FIGS. 3 and 4 are alternative cross-sectional views for thecatheter of FIG. 1.

[0031] FIGS. 5A-5C illustrate use of the catheter of FIG. 1 inperforming nucleic acid transfection within a blood vessel.

[0032]FIG. 6 illustrates a kit constructed in accordance with theprinciples of the present invention.

[0033] FIGS. 7-10 are charts and graphs comparing transfection accordingto the present invention with controls. Porcine VSMCs (FIGS. 7 and 9)and ECs (FIGS. 8 and 10) were transfected for 3 h with naked or liposome(Promega Tfx-50) complexed luciferase DNA (n=12) and luciferase activityin cell lysates was determined after 48 h at 37° C. (FIGS. 7 and 8).Parallel adherent cell counts were performed at baseline (time 0) and at3, 18 and 48 h after transfection (FIGS. 9 and 10). Where applicableultrasonic energy (1 MHz, CW, 0.4 W/cm², 60 s) was applied for 30minutes into the 3 h transfection period. Asterisks indicate significantdifferences between control and ultrasound-exposed cells (p<0.05).

[0034]FIG. 11 compares cumulative mitosis in two wells of subconfluentporcine VSMCs which were observed concurrently by TLVM for 48 h and thecumulative rate of mitosis was analyzed (n=3). One well was exposed toultrasound (1 MHz, CW, 0.4 W/cm², 60 s) prior to filming. Asterisksindicate significant differences between control and ultrasound-exposedcells (p<0.05).

DETAILED DESCRIPTION OF THE INVENTION

[0035] The nucleic acids delivered by the methods and devices of thepresent invention will comprise nucleic acid molecules in a formsuitable for uptake into target cells within a host tissue, usuallysmooth muscle cells lining the blood vessels. The nucleic acids willusually be in the form of bare DNA or RNA molecules, where the moleculesmay comprise one or more structural genes, one or more regulatory genes,antisense strands, strands capable of triplex formation, or the like.Commonly, such nucleic acid constructs will include at least onestructural gene under the transcriptional and translational control of asuitable regulatory region. Optionally, but not necessarily, the nucleicacids may be incorporated in a viral, plasmid, or liposome vesicledelivery vehicle to improve transfection efficiency.

[0036] If viral delivery vehicles are employed, they may comprise viralvectors, such as retroviruses, adenoviruses, and adeno-associatedviruses, which have been inactivated to prevent self-replication butwhich maintain the native viral ability to bind a target host cell,deliver genetic material into the cytoplasm of the target host cell, andpromote expression of structural or other genes which have beenincorporated in the particle. Suitable retrovirus vectors for mediatedgene transfer are described in Kahn et al. (1992) Circ. Res.71:1508-1517, the disclosure of which is incorporated herein byreference. A suitable adenovirus gene delivery is described in Rosenfeldet al. (1991) Science 252:431-434, the disclosure of which isincorporated herein by reference. Both retroviral and adenovirusdelivery systems are described in Friedman (1989) Science 244:1275-1281,the disclosure of which is also incorporated herein by reference. Thenucleic acids may preferably be present in a lipid delivery vehiclewhich enhances delivery of the genes to target smooth muscle cellswithin the vascular epithelia or elsewhere. Transfection in a lipiddelivery vehicle is often referred to as “lipofection.” Such deliveryvesicles may be in the form of a liposome where an outer lipid bilayersurrounds and encapsulates the nucleic acid materials. Alternatively,the nucleic complexes may be in the form of a nucleic acid-lipiddispersion, nucleic acid-lipid emulsion, or other combination. Inparticular, the complexes may comprise liposomal transfection vesicles,including both anionic and cationic liposomal constructs. The use ofanionic liposomes requires that the nucleic acids be entrapped withinthe liposome. Cationic liposomes do not require nucleic acid entrapmentand instead may be formed by simple mixing of the nucleic acids andliposomes. The cationic liposomes avidly bind to the negatively chargednucleic acid molecules, including both DNA and RNA, to yield complexeswhich give reasonable transfection efficiency in many cell types. See,Farhood et al. (1992) Biochem. Biophys. Acta. 1111:239-246, thedisclosure of which is incorporated herein by reference. A particularlypreferred material for forming liposomal vesicles is lipofection whichis composed of an equimolar mixture of dioleylphosphatidyl ethanolamine(DOPE) and dioleyloxypropyl-triethylammonium (DOTMA), as described inFelgner and Ringold (1989) Nature 337:387-388, the disclosure of whichis incorporated herein by reference.

[0037] It is also possible to combine these two types of deliverysystems. For example, Kahn et al. (1992), supra., teaches that aretrovirus vector may be combined in a cationic DEAE-dextran vesicle tofurther enhance transformation efficiency. It is also possible toincorporate nuclear proteins into viral and/or liposomal deliveryvesicles to even further improve transfection efficiencies. See, Kanedaet al. (1989) Science 243:375-378, the disclosure of which isincorporated herein by reference.

[0038] The nucleic acids will usually be incorporated into a suitablecarrier to facilitate delivery and release, into the blood vesselsaccording to the present invention. The carriers will usually be liquidsor low viscosity gels, where the nucleic acids will be dissolved,suspended, or otherwise combined in the carrier so that the combinationmay be delivered through the catheter and/or carried by the catheter andreleased intravascularly at the treatment site. Alternatively, thenucleic acids may be provided in a dry or solid form and coated onto orotherwise carried by the catheter or the vibrational surface. Anexemplary catheter 10 suitable for use in the methods of the presentinvention is illustrated in FIGS. 1 and 2. The catheter 10 comprises acatheter body 12 having a proximal end 14, a distal end 16, and avibrational interface surface 18 near the distal end. The vibrationalinterface surface 18 comprises a piezoelectric ceramic 20 disposedbetween an insulating layer 22 and an aluminum shim 24. An air gap 26 isbehind the shim, and the transducer assembly is suitable for a highfrequency oscillation. The catheter further includes a central lumen 28to enable the catheter to be delivered over a guidewire in aconventional manner. The catheter further includes a pair of axiallyspaced-apart balloons 30 and 32 on either side of the vibrationalinterface surface 18. The balloons 30 and 32 may be inflated via aninflation port 33 on a proximal hub 23 secured to the proximal end 14 ofthe catheter body 12. The proximal hub also includes an infusion port 34which can deliver an infusate, usually comprising the nucleic acids tobe delivered, through a port 35 between balloons 30 and 32 and proximatethe vibrational interface surface 18. The hub further includes wires 36which permit the transducer 24 to be connected to a suitable driver,e.g. a commercially available signal generator and power amplifiercapable of exciting the transducer within the target frequency rangesand intensities.

[0039] While a single transducer 24 is illustrated in FIGS. 1 and 2, itwill frequently be desirable to provide multiple transducers 20, asillustrated in FIG. 3, or a circularly symmetric transducer 40, asillustrated in FIG. 4. As illustrated in FIG. 3, a plurality oftransducers 20 could be circumferentially spaced-apart about theexterior of the catheter body 12. In this way, energy can be transmittedradially outwardly in multiple directions at once. In order to enhancethe uniformity of the treatment, the catheter could optionally berotated while the energy is being delivered. In order to further enhancethe uniformity of ultrasonic energy being radiated outwardly, themultiple transducer embodiments can be driven by a multiplexd powersource. To still further enhance the uniformity of ultrasonic energybeing delivered, a piezoelectric transducer 40 can be formed in acylindrical geometry, as illustrated in FIG. 4. The transducer ceramic40 can be driven by inner and outer electrodes 42 and 44, and the outerelectrodes coated by a thin insulating layer 46. A transducer ceramiccan be supported on a suitable cylinder, such as an aluminum cylinder48, and for high frequency operation an air gap 50 may be provided. Thetransducer can be mounted symmetrically about catheter body 52 having aconventional guidewire lumen 54. In all of the above cases, thedimensions will depend in large part on the frequency of operation aswell as the catheter size. The width of the transducers will typicallybe in the range from 0.1 mm to 6 mm, usually from 0.5 mm to 3 mm. Thelength of the transducer may vary from 1 mm to 2 or more cm, with thelength being primarily limited by loss of flexibility of the distal endof the catheter. Multiple transducer elements could also be providedalong the length of the catheter, i.e. being axially spaced-apart. Othertransducer designs may be employed, such as those disclosed in thecopending applications cited above.

[0040] Catheter 10 may be used to deliver nucleic acids to a targetregion within a blood vessel BV, as illustrated in FIGS. 5A-5C. Thecatheter 10 is intravascularly introduced to the target region in aconventional manner. Once at the desired target region, as shown in FIG.5A, the balloons 30 and 32 will be inflated, as illustrated in FIG. 5B,to define an isolated region R within the blood vessel lumen. A suitableliquid medium containing the nucleic acids to be delivered can then beintroduced via port 34 and orifice 35 into the isolated region R until adesired concentration of the nucleic acid is achieved. Optionally, thenucleic acid may be replenished and/or recirculated within the region ifdesired. After sufficient nucleic acid has been introduced, thevibrating interface surface 18 is actuated in order to transmitultrasonic energy through the medium into the blood vessel wall toenhance uptake of the nucleic acids, as illustrated in FIG. 5C. Thevibrational energy is delivered within the frequency ranges and at theintensities described above, typically for periods of time from 10 sec.to 10 min., usually from 20 sec. to 3 min.

[0041] Referring now to FIG. 6, the catheters 10 of the presentinvention will usually be packaged in kits. In addition to the catheter10, such kits will include at least instructions for use 50 (IFU). Thecatheter and instructions for use will usually be packaged togetherwithin a single enclosure, such as a pouch, tray, box, tube, or the like52. At least some of the components may be sterilized within thecontainer. Instructions for use 50 will set forth any of the methodsdescribed above.

[0042] The following examples are offered by way of illustration, not byway of limitation.

EXPERIMENTAL

[0043] 1. Methods

[0044] A. Cell Culture

[0045] Explant-derived porcine medial vascular smooth muscle cells(VSMCs) and enzyme-dispersed luminal endothelial cells (ECs) wereobtained from the thoracic aorta of Yorkshire White cross pigs agedunder 6 months and cultured on gelatin-coated tissue culture flasks(Costar) in Dulbecco's Modified Eagle Medium (DMEM) containing 10%porcine serum; EC cultures were supplemented with EC growth factor (20μg/ml; Sigma) and heparin (90 μg/ml; Sigma).

[0046] B. Transfection Conditions

[0047] All transfections were for 3 h at 37° C. in 24-well plates withcells at 60-70% confluence, and were stopped by dilution with 1 ml offresh culture medium. Naked DNA transfections were carried out in 200 μlDMEM containing 10% porcine serum and 7.5 μg/ml luciferase plasmid DNA(pGL3, Promega) per well. Lipofections were carried out using PromegaTfx-50 (which contains DOPE), according to conditions optimized forVSMCs (200 μl DMEM containing 10% porcine serum; DNA:lipid charge ratioof 4:1; 7.5 μg/ml final DNA concentration) and ECs (200 μl serum-freeDMEM; DNA:lipid charge ration 3:1; 5 μg/ml final DNA concentration).

[0048] Where applicable, ultrasound exposure (USE) was performed 30 min.into the transfection period using a 10 mm diameter air-backedpiezoelectric flat plate ceramic transducer activated to producecontinuous wave (CW) 1 MHz ultrasound at 0.4 W/cm² using amultifunctional signal generator (DS 345, Stanford Research Systems)working through a Krohn-Hite 7500 power amplifier, monitoredcontinuously using an oscilloscope (TDS 220, Tektronix).. The 24-wellplates were suspended in a 2 cm-deep polystyrene water bath at 37° C.during USE, which was performed for 60 s with the transducer within thetransfection medium 2 mm above the cell monolayer. This level of USEcaused only minor acute damage to the cell monolayer and had no effecton naked or liposome-complexed plasmid DNA integrity as accessed byagarose gel electrophoresis (data not shown). Temperature was recordedcontinuously using a custom-built computerized probe placed adjacent tothe ultrasound transducer. A 10 mm diameter heating probe wasconstructed in-house and used to mimic the rate of rise and finaltemperature achieved during USE.

[0049] C. Assays for Luciferase Activity, Adherent Cell Number andValidity

[0050] Luciferase activity in VSMC and EC lysates 48 h aftertransfection was measured using the GenGlow kit and 1253 Luminometer(BioOrbit) and expressed as light units per microgram total cell protein(assayed in parallel using the Bradford method (BioRad)). Parallel wellswere trypsinised at 0, 3, 18 and 48 h after treatment. Cell counts andviability were assayed by Coulter™ counter and FACS analysis ofpropidium-iodide and fluorescein diacetate exclusion.

[0051] D. TimeLapse Video Microscopy (TLVM)

[0052] Identically seeded subconfluent VSMCs in 24-well plates wereobserved by TLVM using a Leitz DM 1RB inverted microscope (Leica UK Ltd)within a 37° C. environment chamber. One frame of a high-power field wasrecorded every 2.4 min. at for 48 h beginning 3 h after USE (whereapplicable) using a monochrome video camera (Sony), Super-VHS videorecorder (Panasonic) and a BAC900 animation controller (EOS electronicsAV Ltd, Barry, UK). A mitotic event was recorded when 2 daughter cellsappeared from a single dividing cell. An apoptotic event was recordedwhen an individual cell underwent the typical morphological changes ofmembrane blebbing, cytoplasmic shrinkage, nuclear condensation anddislodgement.

[0053] E. Statistical Analysis

[0054] All data are presented as mean ±SEM. Treatments were comparedusing the Friedman ANOVA test, and the Wilcoxon signed rank test forpost hoc comparisons. Values were considered to be significantlydifferent if p<0.05, applying the Bonferroni correction for multiplecomparisons were appropriate. The n numbers quoted refer to the numberof separate experiments; on each occasion each treatment was performedin triplicate wells.

[0055] F. In Vivo Tests

[0056] Catheter balloon mounted 3.5×15 mm cationic Matrix HI Biodiv Yio™stents (BioCompatibles) were dipped in a saline solution containingpGL3-luciferase plasmid (Promega) at 3 mg/ml for 12 hours at roomtemperature and then air dried. Mean stent loading was 5 μg per stent.

[0057] For all stent placement procedures, pigs were sedated withKetamine and intubated, ventilated with oxygen, and anesthetized withisoflurane. Femoral artery access was obtained, and an 8 Fr guidingcatheter was advanced to the left coronary ostium. ECG and bloodpressure were continuously monitored during the study. Underangiographic guidance, the plasmid coated stent was placed in the LADand RCA coronary arteries in each animal. The catheter balloon wasinflated to reach a balloon to artery dilation ration of 1.2 to achievestent implantation.

[0058] After each stent deployment, a 5 Fr Sonotherapy™ catheter(PharmaSonics) was advanced, under angiographic guidance, so that thetransducer elements were positioned within the stent. Stents in the LADreceived a 5 minute ultrasound exposure followed by a 5 mm pullback ofthe catheter then a second 5 minute ultrasound exposure. The ultrasoundconditions were 1 MHz, 3.5 Ml and 0.15% DC. Stents in the RCA receivedthe catheter with the same timing and pullback sequence, but with noultrasound and served as the sham control. At the completion of theultrasound and sham treatments, catheters were removed, the femoralartery ligated and the wound closed.

[0059] Four days after the stent procedure, the animals were euthanized,the hearts were quickly removed, and arterial sections from the LAD andRCE containing the stents were explanted. An additional arterial sectionfrom the LCX was also taken from each animal to enable measurement ofthe endogenous luciferase activity in the arterial tissue. Afterremoving the stent struts from the arterial sections, the tissue washomogenized then centrifuged and a soluble fraction containing theluciferase was obtained. Samples were assayed for the luciferase geneproduct with the GenGlow kit (Labtech International) and measured in a1253 Luminometer BioOrbit). Luciferase activity was expressed in lightunits per 3 mm of vessel length.

[0060] 2. Results

[0061] Luciferase activity was barely detectable in VSMC lysates 48 hafter transfection with naked plasmid alone (0.4±0.2 LU/μg), but was7.5-fold higher in parallel wells exposed to 1 MHz ultrasound (3.0±2.0LU/μg; n=12; p<0.02 cf naked DNA alone), equivalent to 11% of thatachieved following optimal lipofection alone (27.6±6.9 LU/μg) (FIG. 7).USE during lipofection further enhanced reporter gene expression, byalmost 3-fold (72.8±17 LU/μg; n=12; p<0.002 cf lipofection alone) (FIG.7). The temperature of the culture medium increased progressively duringUSE, reaching 14±1° C. above baseline after 60 s. To exclude thepossibility that ultrasound-induced heating may be responsible for theobserved effects on reporter gene expression, VSMCs were exposed to anidentical rate and final temperature rise over 60 s in the absence of 1MHz ultrasound. No effect on luciferase activity in VSMC lysates after48 h was observed (FIG. 7).

[0062] Luciferase activity was almost undetectable in EC lysates 48 hafter transfection with naked DNA (0.7±0.1 LU/μg) but, in contrast tothe results with VSMCs, was not enhanced by adjunctive USE; (1.2±0.2LU/μg; n=4; p=NS of naked DNA alone) (FIG. 8). USE during lipofection,however, enhanced reporter gene expression in ECs by more than 3-fold,from 17.7±1.1 to 57.8±20.2 LU/μg (n=4; p<0.04).

[0063] USE had no significant effect on adherent cell number at 3 h butwas associated with much smaller subsequent increases compared witheither untreated control wells or those exposed to a temperature risealone (FIG. 9). This effect was not observed in ultrasound-exposed EC,which increased in number in identical fashion to control cells (FIG.10). Adherent VSMC and EC viability was identical in control,heat-exposed and 1 MHz-treated wells and remained unchanged throughout(p=NS for each treatment and at all timepoints, data not shown). We didnot observe an excess of detached VSMCs in the culture medium followingUSE, either by eye or by performing cell counts on the culture mediumitself (data not shown). TVLM analysis of identically prepared, randomlycycling, subconfluent VSMCs showed that ultrasound exposuresignificantly reduced the rate of mitosis (FIG. 11). In contrast,ultrasound had no effect on the rate of apoptosis in the same cultures(cumulative percent apoptosis in control wells; 9.9±4.2% at 24 h,11.6±5.0% at 48 h. In ultrasound-exposed wells; 4.8±4.3% a 24 h,7.6±5.2% at 48 h; n=3; p=NS for all comparisons).

[0064] The following Table is the matched data for each animal showingthe fold enhancement of luciferase activity normalized to the luciferasebackground from the LCx (luc. act. RCA/LCx & LAD/LCx). The resultsindicate about a four-fold enhancement with a significant p value whenultrasound was applied. TABLE Fold Enhancement Animal No. RCA LAD 1 2.257.05 2 1.86 8.14 3 3.19 5.54 4 1.76 4.97 5 2.31 5.88 Mean: 2.27 6.32S.D.: 0.56 1.27 p:  0.004

[0065] 3. Discussion

[0066] In the present study we demonstrate that adjunctive USE enhancesreporter gene expression following optimal naked DNA and/or li8pofectionof primary vascular smooth muscle cells. A number of recent reports haveshown that ultrasound also enhances reporter gene expression followingtransfection of non-vascular, mainly immortalized, cells in vitro,including human prostate cancer (Tata et al. (1997) Biochem. Biophys.Res. Comm. 234;64-67), chondrocyte (Greenleaf et al. (1998) UltrasoundMed. Biol. 24:587-595), Chinese Hamster Ovary (Bao et al. (1997)Ultrasound Med. Biol. 23:953-959), and HeLa cell lines (Unger et al.(1997) Invest. Radiol. 32:723-727), primary rat fibroblasts andchondrocytes (Kim et al. (1996) Hum. Gene. Ther. 7:1339-1346), and mouseNIH/3T3 and mammary tumor cell lines (Unger et al. (1997) Invest.Radiol. 32:723-727). Transfection rates of up to 15% of survivingimmortalized human chondrocytes using naked DNA have been reportedfollowing exposure to CW 1 MHz ultrasound, and two to 1000-foldenhancements in lipofection efficiency have been reported in a number ofimmortalized cell lines, in each case independent of heat. The three to7.5-fold enhancements recorded herein probably underestimate the effectsof ultrasound for several reasons. First, USE was performed from aboveto minimize standing wave formation resulting from reflection atfluid/air and plastic/air interfaces. This constrained transducer designsuch that only one-third of each cell monolayer was covered by thetransducer. Secondly, the choice of ultrasound parameters may not havebeen optimal. We used 1 MHz ultrasound at less than 1 W/cm² as thiscorresponds to the mean output of diagnostic transducers and isclinically safe (Henderson et al. (1995) Ultrasound Med. Biol.21:699-705; Barnett et al. (1996) Ultrasound Med. Biol. 24:i-xv, S1-58).Furthermore, USE at this level had no effect on DNA integrity orvascular cell viability in vitro. Additionally, the small number ofcells physically dislodged acutely during USE may have been those mostlikely to have been transfected. These cells were lost to analysis underthese tissue culture conditions, although this situation may not pertainto VSMCs and ECs within the intact vessel wall in vivo.

[0067] Low intensity ultrasound requires pre-formed microbubbles ornucleation sites to generate cavitation, and these conditions certainlyexist in the non-degassed culture media used in our experiments. Theeffects of ultrasound in vitro may be further enhanced in the presenceof additional microbubbles in the form of the echocontrast agentAlbunex™, and naked DNA transfection rates approaching those achievedwith lipofection have been reported (Tata et al. (1997) Biochem.Biophys. Res. Comm. 234:64-67). There is relatively little evidence forthe existence of microbubbles or cavitation nuclei, however, in blood.Thus, methods according to the present invention which combine lowintensity USE and local delivery of DNA mixed with (or even within)microbubbles would be useful not only to focus but also restrict genedelivery to a desired target site in a blood vessel.

[0068] The results in animals demonstrate that ultrasound can enhanceintra-arterial gene or drug delivery. The method entailed the use of aDNA coated stent and a Sonotherapy catheter. The same ultrasoundconditions were used for DNA delivery that have been found to inhibitintimal hyperplasia. See, for example, U.S. Pat. No. 6,210,393, havingcommon inventorship with the present application.

[0069] While the above is a complete description of the preferredembodiments of the invention, various alternatives, modifications, andequivalents may be used. Therefore, the above description should not betaken as limiting the scope of the invention which is defined by theappended claims.

What is claimed is:
 1. A method for intravascular nucleic acid delivery,said method comprising: providing a flexible catheter having avibrational transducer disposed near its distal end; intravascularlypositioning the distal end of the catheter at a target region within ablood vessel; delivering nucleic acids to vascular smooth muscle cellswhich line a wall of the blood vessel; and energizing the transducer todeliver vibrational energy to the wall at a frequency and intensityselected to enhance uptake of the nucleic acids by the smooth musclecells, wherein expression of the nucleic acids in the cells exposed tothe vibrational energy is at least two-fold greater than in comparablecells not exposed to vibrational energy.
 2. A method as in claim 1,wherein the expression of the nucleic acids is at least four-foldgreater than in comparable cells not exposed to vibrational enegy.
 3. Amethod as in claim 1, wherein the vibratory energy is at a frequency inthe range from 1 kHz to 10 MHz.
 4. A method as in claim 3, wherein thevibratory energy has an intensity in the range from 0.01 W/cm² to 100W/cm².
 5. A method as in claim 4, wherein the vibratory energy isdelivered with a duty cycle in the range from 1% to 100%.
 6. A method asin claim 5, wherein the vibratory energy is applied for a cumulativetreatment time in the range from 10 seconds to 900 seconds.
 7. A methodas in claim 1, wherein the interface surface directly contacts the bloodvessel wall within the target region.
 8. A method as in claim 1, whereinthe interface surface is spaced-apart from the blood vessel wall,wherein ultrasonic energy is transmitted through a liquid mediumcontaining the nucleic acids disposed between the interface surface andthe wall.
 9. A method as in claim 1, wherein the vibrational excitingstep comprises vibrating the surface in a radial direction.
 10. A methodas in claim 1, wherein the vibrational exciting step comprises vibratingthe surface in an axial direction.
 11. A method as in claim 1, furthercomprising expanding a pair of axially spaced-apart balloons disposed oneither side of the vibrational surface to localize a solution containingthe nucleic acids as the surface is vibrated.
 12. A method as in claim1, wherein the nucleic acids are delivered through the flexiblecatheter.
 13. A method as in claim 1, wherein the nucleic acids areselected from the group consisting of genes, gene fragments, sensepolynucleotides, anti-sense polynucleotides, and oligonucleotides.
 14. Amethod as in claim 13, wherein the nucleic acids encode an angiogenicfactor eNOS, TIMP, or p21.
 15. A method as in claim 1, wherein thenucleic acids are delivered after the target region has been treated toenlarge or remove an occlusion.
 16. A method as in claim 15, wherein theprior treatment is selected from the group consisting of angioplasty,atherectomy, and stenting.
 17. A kit comprising: a catheter having avibratory interface surface; and instructions for use setting forth amethod according to claim
 1. 18. A system comprising: a catheter havinga vibratory interface surface; and a nucleic acid reagent which can beintravascularly delivered by the catheter.